ProjectSummary Bacterialpathogenscanpersistoverthelifetimeoftheirhostinagrowthrestricted,dormantstate.Pathogens likeClostridiumdifficile,thatcansurviveassporesareespeciallyresistanttoantibiotics.C.difficileisestimated tocosttheUShealthcaresystemmorethanonebilliondollarsperyear.Understandinghowbacteriaregulate dormancyisessentialtoourabilitytotreatthesekindsofinfection.Tobecomequiescent,cellsmustshutdown proteinsynthesis.Post-translationalmodificationsoffactorsinvolvedintranslationarelikelytoplayarolein thisregulation.TheproposedworkaimstodeterminetheroleofelongationfactorG(EF-G)phosphorylationin regulatingribosomerecyclingduringdormancy.EF-Gisstablyphosphorylatedinregionsthatinteractwith ribosomerecyclingfactor(RRF)duringstationaryphaseinmanybacteria.EF-Gisalsoknowntobestably phosphorylatedinthemodelspore-formerBacillussubtilis.Usingbiochemicalapproaches,wewillinvestigate theeffectsoftheseconservedmodificationsonribosomerecycling,andontheGTPaseactivityofEF-G.The roleofEF-Gintherecyclingofribosomessequesteredbyhibernationfactorswillalsobedetermined.Using singlemoleculefluorescenceenergytransfer(smFRET)approaches,wewillinvestigatetheshort-lived interactionsofP~EF-GwithRRFonindividualribosomesthathaveterminatedtranslation.Finally,sincelittleis knownabouthowtranslationisinhibitedinsporeswewillprofiletheassociationofribosomalsubunitsandtheir activityoverthecourseofsporedevelopmentandasthesporegerminates.Wewillalsoidentifyphosphosites onEF-Ginthesporeanddeterminetheeffectsofthesemodificationsontherecyclingandelongationactivity ofEF-Ginvitro.Thesestudieswillimpactourunderstandingofribosomerecyclingandhowphosphorylation canbeusedtoregulatedormancy.